Rheologic and Vascular Modulators in Sickle Vaso-occlusion ABSTRACT Sickle cell disease (SCD) is characterized by episodes of painful vaso-occlusive crisis and multiple organ damage, severely compromising the quality of life in the affected individuals. Tissue hypoxia is of primary importance to the pathogenesis of SCD and its attendant complications. Chronic hypoxia is a predictable consequence of abnormal red cell rheology, sickling, hemolytic anemia and recurring vaso-occlusive episodes. In SCD, chronic tissue hypoxia and oxidative stress are likely to activate hypoxia inducible factor (HIF)-11, a key protein regulating cellular responses to tissue hypoxia. HIF-11 activation and its potential stabilization in SCD (supported by our preliminary observation in transgenic sickle mice) will have both adverse (altered vascular tone) and beneficial (cytoprotective) consequences on microvascular regulation. We hypothesize that HIF-11 expression will be dependent not only on tissue oxygen tension, but also on nitric oxide (NO) bioavailability, carbon monoxide (CO) generation and increased oxidative stress (reperfusion injury). Also, the extent and duration of NO and CO signaling modulated by hemolytic rate and oxidant generation will regulate HIF-11 expression. HIF-11 up-regulates a variety of genes including those for vasoactive stimuli and it also confers protection against reperfusion injury although the role of this transcription factor remains unexplored in SCD. The objective of the proposed in vivo studies is to delineate the relative contribution of these likely mechanisms. In this resubmission, we will focus on the following interrelated aspects: i) examine the factors and mechanisms involved in the regulation of HIF-11 expression in SCD, ii) delineate the role of HIF-11 using HIF-11-deficient sickle mice, and ii) investigate relative roles of hemolytic rate and oxidative stress in HIF-1 activation. We believe that the proposed studies constitute the first attempt to understand these aspects under in vivo conditions. Under Specific Aim 1, we will test the hypothesis that intravascular sickling, tissue oxygen tension, reduced NO bioavailability and increased CO production will regulate HIF-11 expression and affect microvascular hemodynamics. Our preliminary studies show that activation of HIF-11 in transgenic sickle is associated with the induction of HO-1 and vascular endothelial growth factor (VEGF), a surrogate marker for HIF-11. Moreover, we show that hypoxia induces marked activation of HIF-11 in vascular endothelial cells. Under Specific Aim 2, we will test the hypothesis that, in SCD, HIF-11 activation will contribute to altered vascular tone and reactivity and confer cytoprotection against reperfusion injury by modulating expression of vasoactive/ cytoprotective molecules (e.g., HO-1). We will investigate this aspect using bone marrow transplantation from sickle mice into mice partially deficient in HIF-11, and by super induction of HIF-11. Under Specific Aim 3, we will test the hypothesis that, in SCD, hemolysis and oxidative stress have distinct effects on HIF-11 expression and microvascular regulation via NO-dependent mechanism. To explore our hypotheses, we will use state-of-the art transgenic sickle mouse models and mice deficient in HIF-11, and combine intravital techniques with cellular and molecular analyses. The proposed in vivo studies are expected to provide insights into the adverse and beneficial effects of HIF-11 and NO in SCD, which may lead to new therapeutic strategies. PUBLIC HEALTH RELEVANCE: Sickle cell disease (SCD) is characterized by episodes of painful vaso-occlusive crisis, multiple organ damage, and severely compromising quality of life in the affected individuals. Among African-Americans, sickle cell disease occurs at a rate of 1 per 400 births. Red cell sickling in small-diameter blood vessels is considered the ultimate event in the pathophysiology of vaso-occlusion (vessel blockage). In addition, SCD is characterized by tissue hypoxia that may trigger activation of adaptive mechanisms such as activation of hypoxia-inducible factor-11 (HIF-11), while persistent hemolysis and oxidative stress will impair nitric oxide (NO) bioavailability and depletion of NO substrate, arginine. The proposed exploratory studies will focus on the role HIF-11 and NO in SCD. A better understanding of the role of in HIF-11 and NO and their interactions will allow us to design future studies with therapeutic implications.